【格机格机】内毒素屏蔽和低内毒素恢复（LER）Question and Answers to Endotoxin ...

清歌一曲月如霜

屏蔽内毒素和低内毒素恢复是当前经常讨论的话题涉及到药品的质量。由于内毒素掩蔽和低内毒素回收九月（LER）结束的研讨会的一部分，参加者有相当多的问题。扬声器，约翰内斯·赖克在研讨会结束时回答了这些问题。下面你会发现这些问题和答案的摘要。
“下面的问题和答案都在研讨会提出的观点的总结。所提供的答案不具有约束力，只起到指导。
做一次相关的内毒素回收的研究必须为所有的配方进行？
是的，这是不够的仅仅是测试单一物质组或制剂类，由于内毒素掩蔽取决于许多不同的因素。例如，不同的pH值的设置可能显著影响掩蔽其它相同条件下进行。
“测试的干扰”和“内毒素屏蔽”之间的区别是什么？
这两个现象有不同的底层机制。万一“测试干扰”，该检测系统的/酶反应直接感到不安的环境条件。例如，蛋白酶抑制剂可妨碍检测的酶，从而导致假阴性结果。
另一方面，如果“内毒素掩蔽”的发生，要被检测的内毒素本身是由环境条件的影响。这意味着该内毒素的活性形式（超分子结构）发生了变化，从而降低检测到的活动。酶检测反应但是不阻碍在这种情况下。在实践中，可能同时发生这两种现象。
你怎么能干扰和屏蔽之间的区别？
作为干扰是与时间无关的和掩蔽是时间依赖性的，依赖于时间的实验，需要在除了阳性对照。这意味着它们的回收率随时间降低，如果掩蔽发生。到的现象区分开来，具有足够高的峰值浓度，必须选择用于未稀释的样品稀释后用于依次排除测试的干扰。
多长时间，并在其孵化温度应掩蔽实验进行？
孵化的长度是高度依赖于各自取样。甲显著减少内毒素的回收率（<50％）的已在某些情况下被观察到，几分钟后已经，例如30分钟。在另一方面，也出现了情况后掩蔽几天才被发现，如5天。
培养温度应选择为匹配其通常发生在其处理过程中样品的环境温度。
它的值作为起点的屏蔽动力学研究？
在LRW（LAL试剂的水）的内毒素的回收率可以被用作初始值（100％）。
其中浓度应在秒杀毒素有哪些？
最佳尖峰浓度依赖于待分析样品和应调整至内毒素限制待检测和微血管密度（最大有效稀释）。低浓度的峰值，如<10 EU / ml时，甚至可能发生在纯LRW不利的行为。
你如何确保LRW和/或样品管不掩盖内毒素？
排除毒素掩蔽在LRW或通过该样品管，内毒素尖峰（在单个样品管）应连同随时间的掩蔽样品进行监测。
其中测试系统可用于检测？
所有常见的检测系统，可用于进行内毒素掩蔽的实验，如鲎（比浊，显色等），重组体的检测系统或单核细胞活化试验（MAT）。
这办法观察屏蔽之后采取？
观察掩蔽后，所使用的测试系统的优化，例如相对于样品制备，或者更换一个替代方法是必要的。
什么是解蔽的原则？
假定内毒素的可检测性依赖于它的超分子结构。这种结构可呈现各种聚合形式，因而不同的活动取决于环境条件。在掩蔽的过程中，内毒素的结构被转换成不可检测的状态下，如单体。返回掩蔽的内毒素进入激活状态，环境条件来选择在该转移的平衡背面朝向“自组织”成活性聚合形式的一种方式“。



Masking of Endotoxins and LOW Endotoxin Recovery is a currently often discussed topic related to the quality of pharmaceutical products. As part of the webinar on Endotoxin Masking and Low Endotoxin Recovery (LER) end of September the participants had a number of questions. The speaker, Johannes Reich, answered these questions at the end of the webinar. Following you will find a summary of these questions and answers.
"The following questions and answers are a summary of the points raised in the webinar. The provided answers are not binding and shall only serve for guidance.
Do studies for time-dependent endotoxin recovery have to be conducted for all formulations?
Yes, it is not sufficient to merely test single substance groups or formulation classes, since endotoxin masking depends on numerous different factors. For instance, different pH settings may significantly influence masking under otherwise identical conditions.
What is the difference between "test interference" and "endotoxin masking"?
Both phenomena have different underlying mechanisms. In case of "test interference", the detection system/ the enzymatic reaction is directly disturbed by the ambient conditions. For example, protease inhibitors can impair the detection enzyme and thereby cause a false-negative result.
On the other hand, if "endotoxin masking" occurs, the endotoxin to be detected itself is influenced by the ambient conditions. This means that the active form (supramolecular structure) of the endotoxin has changed, leading to lower activities that are detected. The enzymatic detection reaction is however not inhibited in this case. In practice, both phenomena may occur simultaneously.
How can you differentiate between interference and masking?
As interference is time-independent and masking is time-dependent, time-dependent experiments are required in addition to the positive control. This means that the recovery rate decreases over time, if masking occurs. To differentiate between the phenomena, a sufficiently high spike concentration has to be chosen for the undiluted samples to later dilute for in turn excluding test interferences.
How long and at which incubation temperature shall a masking experiment be conducted?
The length of incubation is highly dependent on the respective sample. A significant decrease of endotoxin recovery (<50%) has been observed in some cases already after a few minutes, e.g. 30 min. On the other hand, there have been cases where masking was only found after several days, e.g. 5 days.
The incubation temperature shall be chosen to match the sample's ambient temperature which commonly occurs in the course of its handling.
Which value is used as start in a masking kinetic study?
The endotoxin recovery in LRW (LAL reagent water) can be used as start value (100%).
Which concentration shall the endotoxin spike have?
The optimum spike concentration depends on the sample to be analyzed and shall be adjusted to the endotoxin limit to be detected and the MVD (maximum valid dilution). Low spike concentrations, e.g. <10 EU/ml, may even happen to behave unfavorably in pure LRW.
How do you ensure that LRW and/or the sample tube do not mask endotoxin?
To exclude endotoxin masking in LRW or by the sample tube, the endotoxin spike (in a single sample tube) shall be monitored together with the masking sample over time.
Which test systems can be used for detection?
All common detection systems can be used for conducting endotoxin masking experiments, e.g. LAL (turbidometric, chromogenic etc.), recombinant detection systems or monocyte activation tests (MAT).
Which measures shall be taken after observing masking?
After observing masking, the optimization of the used test system, e.g. with respect to sample preparation, or the replacement with an alternative method are advised.
What is the principle of demasking?
It is assumed that the detectability of endotoxin depends on its supramolecular structure. This structure may exhibit various aggregation forms and thus different activities depending on the ambient conditions. In the course of masking, the endotoxin structure is converted into an undetectable state, e.g. monomers. To return masked endotoxin into an active state, the ambient conditions have to be chosen in a way that shifts the balance back toward "self organization" into active aggregation forms."

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